Exosymbiotic microbes within fermented pollen provisions are as important for the development of solitary bees as the pollen itself

Abstract Developing bees derive significant benefits from the microbes present within their guts and fermenting pollen provisions. External microbial symbionts (exosymbionts) associated with larval diets may be particularly important for solitary bees that suffer reduced fitness when denied microbe‐colonized pollen. To investigate whether this phenomenon is generalizable across foraging strategy, we examined the effects of exosymbiont presence/absence across two solitary bee species, a pollen specialist and generalist. Larvae from each species were reared on either microbe‐rich natural or microbe‐deficient sterilized pollen provisions allocated by a female forager belonging to their own species (conspecific‐sourced pollen) or that of another species (heterospecific‐sourced pollen). Our results reveal that the presence of pollen‐associated microbes was critical for the survival of both the generalist and specialist larvae, regardless of whether the pollen was sourced from a conspecific or heterospecific forager. Given the positive effects of exosymbiotic microbes for larval fitness, we then examined if the magnitude of this benefit varied based on whether the microbes were provisioned by a conspecific forager (the mother bee) or a heterospecific forager. In this second study, generalist larvae were reared only on microbe‐rich pollen provisions, but importantly, the sources (conspecific versus heterospecific) of the microbes and pollen were experimentally manipulated. Bee fitness metrics indicated that microbial and pollen sourcing both had significant impacts on larval performance, and the effect sizes of each were similar. Moreover, the effects of conspecific‐sourced microbes and conspecific‐sourced pollen were strongly positive, while that of heterospecific‐sourced microbes and heterospecific‐sourced pollen, strongly negative. Our findings imply that not only is the presence of exosymbionts critical for both specialist and generalist solitary bees, but more notably, that the composition of the specific microbial community within larval pollen provisions may be as critical for bee development as the composition of the pollen itself.


| INTRODUC TI ON
Solitary bees, which represent the vast majority of global bee diversity , are among the most important insect pollinators within agricultural and seminatural landscapes (Garibaldi et al., 2013;Klein et al., 2018;. Over the past decade, solitary bee populations have been facing rapid declines (Powney et al., 2019) due to increasing threats from pesticide overuse (Azpiazu et al., 2019), novel diseases (Ravoet et al., 2014), and landscape fragmentation (Kline & Joshi, 2020). Aspects of life history such as pollen specialization (Biesmeijer et al., 2006;Bommarco et al., 2010) and limited foraging range (Greenleaf et al., 2007) render this species-rich group of wild pollinators more susceptible to such risk factors (Burkle et al., 2013;Kremen & Ricketts, 2000;Sgolastra et al., 2019). Along with these well-known stressors, growing evidence suggests that the partnership between solitary bees and their microbial symbionts may play an important role in determining bee fitness. Collectively known as the solitary bee microbiome (Voulgari-Kokota et al., 2019), symbiotic microbes have been shown to perform significant nutritive Gilliam et al., 1984; and protective functions for the developing larvae McFrederick et al., 2012), shaping the overall fitness of these critical pollinators.
The microbiome of larval pollen provisions may be especially critical for the maturation of solitary bees since they have limited opportunities of acquiring microbial symbionts through brood care and/or social interactions with other nestmates . Primarily sourced from the environment (McFrederick et al., 2012Rothman et al., 2019;Voulgari-Kokota et al., 2018), some of these external symbionts (exosymbionts) are thought to be involved with the fermentation and/or preservation of pollen-nectar provisions prior to larval consumption (Gilliam et al., 1984;Lozo et al., 2015;McFrederick et al., 2018;. Empirical data from diet reconstruction studies suggest that the heterotrophic microbes enmeshed within the pollen provisions literally consume and assimilate the resources within plant biomass (i.e., pollen), effectively displacing plant biomass with that of their own Steffan et al., 2017). Because the microbial communities are able to access and consolidate pollen nutrients (amino acids, lipids, and non-structural carbohydrates), these microbes likely serve as conduits for nutrient transfer from pollen to larval biomass, directly influencing the brood outcome among solitary bees (Dharampal, Hetherington, et al., 2020;. In fact, tracing microbial 'fingerprints' using trophic biomarkers suggests that these exosymbionts may represent a direct and dominant source of proteins and lipids for the developing bees, their contribution often exceeding that of pollen itself ).
An individual solitary bee nest, which is provisioned by a single foraging female, contains several discrete brood chambers, each stocked with a one-time supply of pollen, nectar, and sometimes floral oils . The brood chambers host a diverse community of biologically important microbes, including specialized taxa that are reportedly involved with pollen degradation, digestion, and preservation (Cohen et al., 2020;Pimentel et al., 2005;Voulgari-Kokota et al., 2020). While bees that progressively provision their larvae with nutritional resources (e.g., honey and bumble bees) can store the pollen-nectar blend for hours to days prior to larval consumption (Anderson et al., 2014), the nest-stored pollen of massprovisioning solitary bees can undergo fermentation for several weeks (Gilliam et al., 1984). The extended storage duration likely provides opportunities for microbes to proliferate to high abundances (Batra et al., 1973;Miliczky, 1985;Roberts, 1971), and enzymatically transform or the specific microbial community within larval pollen provisions may be as critical for bee development as the composition of the pollen itself.

T A X O N O M Y C L A S S I F I C A T I O N
Community ecology; Entomology; Microbial ecology at least strongly influence the nutritive quality of the provision itself. For larval solitary bees, there appears to be an increased dependence on the resident microbes within the fermenting pollen provision (Voulgari-Kokota, McFrederick, et al., 2019), possibly since pollen provisions in such mass provisioning species tend to ferment the pollen for a much longer period than progressive provisioners .
Based on their foraging strategy, some species of solitary bees are characterized as pollen specialists (oligoleges), foraging on a few related plant species belonging to the same family, whereas others as pollen generalists (polyleges) that have a broader host plant range (Cane & Sipes, 2006). Many plant species are known to host both oligoleges and polyleges, and the pollen collected by oligolectic and polylectic bees can span a spectrum of nutritional quality as determined by its protein content . However, past research indicates that pollen collected by oligoleges is often of lower quality and/ or may contain toxic compounds (Dharampal, Hetherington, et al., 2020;Weiner et al., 2010), making it unfit as the sole source of food for polylectic larvae (Brochu et al., 2020;Cane, 2018;Spear et al., 2016;Vanderplanck et al., 2020). The ability of oligolectic larvae to utilize such low-quality pollen has been attributed to their digestive physiology (Dobson & Peng, 1997;Praz et al., 2008).
However, a previous study utilizing Osmia ribifloris, an Ericaceae specialist, has shown that larvae appear more dependent on the function of the natural microbiota (conspecific microbes) associated with the maternally allocated provisions (conspecific pollen), much more so than the identity of the host plant pollen itself (Dharampal, Hetherington, et al., 2020). This finding suggests that the conspecific microbes embedded within the conspecific pollen, rather than the identity of the pollen per se, play an important role in larval nutrition among oligoleges (Dharampal, Hetherington, et al., 2020;Weiner et al., 2010).
A vast body of literature has documented the presence of diverse exosymbiotic microbes associated with solitary bee species (Christensen et al., 2021;Dew et al., 2020;Gilliam, 1997;Graystock et al., 2017;Keller et al., 2013;McFrederick & Rehan, 2016;Rothman et al., 2020). It has been speculated that these exosymbionts likely perform vital nutritive and defensive functions that strongly influence bee health. For instance, the natural conspecific microbiota within the pollen provisions of oligolectic bees may play a critical role in larval development by enhancing the nutritive value of the low-quality pollen collected by oligolectic foragers (Dharampal, Hetherington, et al., 2020). While the microbiome of solitary bees has received growing attention, the nature and magnitude of the fitness benefit provided by the external microbial symbionts has seldom been empirically quantified and compared across bee foraging strategies.
In this study, we hypothesized that the presence of conspecificsourced microbes would have a greater positive impact on larval fitness than conspecific-sourced pollen, and that the magnitude of this beneficial effect would be larger among oligoleges than polyleges.
To test this hypothesis, we conducted two separate experiments: In the first study (Study 1), we reared larvae of the oligolege,  (Figure 1a). We predicted that larvae would perform best when they had access to both conspecific-sourced pollen and conspecific-sourced microbes, with the effect size of microbes being greater than that of pollen. We also predicted that the beneficial effect of microbe availability would be greater for the oligolege than the polylege.
In a follow-up study (Study 2), we manipulated both the source of pollen (conspecific-sourced pollen versus heterospecific-sourced pollen) and the source of microbes (conspecific-sourced microbes versus heterospecific-sourced microbes) used to reinoculate the pollen. Unlike in Study 1, which tested the effect of microbe presence/absence within pollen provisions, Study 2 examined the effect of microbial sourcing. To these ends, Study 2 included microbes in every pollen provision and explicitly tested whether it mattered for bee larvae if their diets contained microbes from conspecific or heterospecific sources. Simultaneously, the study also tested the effect of pollen sourcing; thus, Study 2 allowed for an examination of the main and interactive effects of microbial and pollen sourcing on bee fitness. For this experiment, O. lignaria larvae were reared only on microbe-rich pollen diets, the pollen, and/or microbes being obtained either from a conspecific (O. lignaria) or a heterospecific source. Heterospecific-sourced pollen and heterospecific-sourced microbes were obtained from another polylectic congener, Osmia cornifrons, which was abundant during the time when the study was conducted. In simultaneously examining the relative importance of pollen and microbe sources for larval performance of O. lignaria, we predicted that larval fitness would be highest when both pollen and microbes were sourced from a conspecific forager, and lowest when sourced from a heterospecific ( Figure 1b).

| Bees and pollen provisions
Wild-collected bees from Washington, Utah, and New York were  (Bosch, 1994). Since Osmia sp. provision larger pollen masses for females than males, and since males are much more abundant, we chose to only use the provisions from male cells for this study (Table S1 and Table S1). Study 1, larval diets for all treatments in Study 2 contained pollenassociated microbes. However, the source of microbes, whether conspecific or heterospecific, was experimentally manipulated.

| Experimental design
To prepare the diet treatments for Study 2, we combined 80%  Table S2).
For both studies, larvae were reared from egg to prepupal stage within sterile 48-well plates based on previously described methods (Dharampal et al., 2018). Separate plates were used for each treatment to minimize the risk of cross-contamination. The weights of the rehydrated sterilized pollen and natural pollen fractions were adjusted such that when combined, the end weight of the reconstituted pollen provision was approximately equal to that of a naturally allocated pro-

| Statistical analyses
Study 1: Separate two-way ANOVAs were conducted to test the impact of main and interactive effects of the independent variables, pollen source (two levels: conspecific; heterospecific) and foraging strategy (two levels: oligolectic; polylectic), on the dependent variables, prepupal biomass and developmental time of larvae reared on natural pollen. Median survival time and distribution was compared across all treatments using the log rank and Gehan-Breslow-Wilcoxon tests. Proportional hazard rate based on time to death for each bee species was modeled using Cox regression analysis (Katz & Hauck, 1993). The end point was set at 25 days, and the covariates in the model included pollen-borne microbes (0 = present; 1 = absent) and source of pollen (0 = conspecific; 1 = heterospecific).

| DISCUSS ION
Two studies were conducted to investigate the importance of microbial exosymbionts for solitary bee development. The first study examined whether the presence or absence of microbial exosymbionts was as important for polyleges as oligoleges. The expectation from Study 1 was that if microbial exosymbionts were truly critical for the development of solitary bee larvae, this effect should be consistent across taxa and across foraging strategies. We also predicted that the magnitude of the effect size of exosymbionts would be stronger among oligolectic larvae. In the second study, the importance of microbial sourcing was examined concurrently with that of pollen sourcing to ascertain the relative importance of each of these factors for brood success. In contrast to Study 1 (which investigated the effect of microbe presence/ absence), all diet treatments in Study 2 contained microbes inoculated within larval pollen provisions. However, the source of the microbes was manipulated, allowing us to compare the effects of having conspecific-sourced microbes (i.e., microbes associated with pollen provision allocated by the mother bee of the same species) versus heterospecific-sourced microbes (i.e., microbes F I G U R E 2 Kaplan-Meier survival plot of (a) Osmia ribifloris and (b) Osmia lignaria across diet treatments. Inset symbols along each survival curve correspond to individual treatments for each bee species. Survival analysis indicates significant differences in the median survival time across all eight treatments (log rank test: χ 2 (7) = 75.87, p < .0001, Breslow-Wilcoxon test: χ 2 (7) = 64.89, p < .0001). Survival distribution indicates significant differences across diet treatments within each species; for (a) Osmia ribifloris, Gehan statistic: p < .001; log rank test: p < .001; and for (b) Osmia lignaria, Gehan statistic: p < .001; log rank test: p < .001. Inset grids next to each survival plot indicate pairwise comparisons of survival distribution (*p < .05) This corroborates and extends the findings of previously published research, which documented the importance of pollen-borne microbes for the development of oligolectic larvae Dharampal, Hetherington, et al., 2020), indicating that the same might be true for polyleges as well. Whether allocated by a conspecific or heterospecific foraging female, provisions that were accompanied by their natural microbiota resulted in high-performing ble prepupal biomass when reared on their own pollen or that of the other species, as long as microbes were present. In contrast, the lack of microbes in pollen provisions led to severe brood failure among both species. When reared on pollen that was devoid of microbes, larvae from both species suffered lowered fitness, regardless of whether they were fed pollen sourced from an adult forager of the same species or from the other. This suggests that for both oligolectic and polylectic solitary bees, microbes present within the pollen provision were likely critical for larval survival, regardless of whether the pollen was provisioned by a conspecific or heterospecific forager. Previous work suggests that this association between pollen-associated microbes and larval health may be attributed to the nutritional symbioses between the two. For instance, trophic reconstruction studies using biomarker-based assays have previously revealed that microbial exosymbionts represent nutritional mutualists and direct prey items that facilitate nutrient transfer from pollen provision to larval bees, dramatically improving brood outcome Dharampal, Hetherington, et al., 2020;. These studies have empirically quantified microbially derived proteins and lipids within bee biomass, reporting that pollen-associated microbes form a dominant source of nutrition for developing larvae. Our study corroborates and extends these findings to include a polylectic species, suggesting that larval reliance on their exosymbionts may be more ubiquitous among solitary bees, regardless of their foraging strategy.
The importance of microbes was also reflected in the survival outcome for both the oligolege and polylege, with the presence of microbes profoundly improving survivorship components. Larval survivorship varied significantly based on treatment type; while 90% of the larvae reared in the presence of pollen-associated microbes reached the prepupal stage, survivorship declined dramatically to 10% among those reared on microbe-deficient diets. Whether reared on conspecific-or heterospecific-sourced pollen, larvae of both species suffered significantly higher mortality when microbes were lacking from their diet. In fact, the worst survivorship outcome for both species was noted among larvae reared on heterospecificsourced pollen without microbes (i.e., O. lignaria on sterilized O. ribifloris pollen, and vice versa), where none of the larvae survived to the prepupal stage. In contrast, survivorship improved significantly among larvae reared on microbe-rich pollen, and was comparable for both heterospecific and conspecific pollen sources. This pattern was consistent for the oligolege as well as the polylege, suggesting that the availability of microbes within larval provisions may have been a stronger predictor of brood survival than pollen source for both types of foragers ( Figure 2). Furthermore, hazard analysis based on larval time to death revealed that, unlike pollen source, the absence of microbes represented a severe and significant risk for larval survival. However, the magnitude of the hazard varied across foraging strategies; the risk of death among oligoleges when reared on sterilized diets increased 27 times compared to 20 times for the polylege.
This indicated that oligoleges are more susceptible to the absence of pollen-borne microbiota, presumably due to increased reliance on nutritional exosymbionts associated with their low-quality conspecific pollen, and this was consistent with earlier findings (Dharampal, Hetherington, et al., 2020). Interestingly, survivorship outcome for both species was unaffected by the source of pollen and neither species showed any significant increase in the risk of death when fed pollen that was heterospecific-sourced instead of conspecificsourced. Taken together, these findings strongly imply that the absence of microbes may have a profound adverse impact on larval performance, and that this effect persists across foraging mode and pollen source.
For the second study, we examined the importance of the source of microbes along with that of forage pollen for the development of Growth rate analysis indicated that larval development among polyleges was strongly impacted by the source of pollen and microbes afforded in their diet. Although all larvae had comparable weights at the start of the study, larval biomass began to show significant differences as early as day 5. Over the course of 10 days, the disparity between larvae that received the right microbes and right pollen (i.e., conspecific foraging) versus larvae that received the wrong microbes and wrong pollen (i.e., heterospecific foraging) increased markedly (Figure 3). This implied that for developing bees, the symbioses with their microbial partners were most beneficial when provisions were sourced from a conspecific female paired with the natural conspecific microbiota. Analyses of microbial and pollen sourcing indicated that both the source of pollen and that of the microbes were significant drivers of bee fitness (Figure 4). Furthermore, the impact of each was almost identical, suggesting that the microbial community in a pollen provision was just as important for bee development as the pollen itself.
In manipulating the source of microbes within larval diet, we observed that microbial sourcing had a large impact on larval fitness when pollen was sourced from a heterospecific forager, but not from that of a conspecific. While larvae reared on heterospecific-sourced pollen along with the innate heterospecific-sourced microbes suffered a marked decline in fitness components, those consuming heterospecific-sourced pollen inoculated with conspecific-sourced microbes showed a significant increase in biomass. This implied that for larvae consuming the 'wrong' pollen, replacing the 'wrong' heterospecific-sourced microbes with the 'right' conspecific-sourced ones may have had a strong positive effect on larval health. One explanation for these findings based on previously published studies is that conspecific-sourced microbes likely perform important nutritive functions, such as pollen fermentation and nutrient transfer Voulgari-Kokota, McFrederick, et al., 2019), enhancing the accessibility of nutrients within heterospecific-sourced pollen. Interestingly, the compensatory effects of conspecificsourced microbes did not extend to conspecific-sourced pollen.
Biomass of larvae reared on conspecific-sourced pollen remained comparable among treatments regardless of whether microbes were sourced from a conspecific or heterospecific. The minimal impact of microbial sourcing indicated that larvae may be physiologically  . For instance, as an Ericaceae specialist with a narrow host plant range, O. ribifloris is likely to acquire a distinct community of microbes compared to the generalist, O. lignaria, that forages on a broader diversity of orchard trees (Rothman et al., 2020). Such differences in host plant preferences likely expose the two species to specific microbial taxa which possess specialized functional adaptations to the respective pollen types (e.g., detoxification of secondary metabolites). Additionally, the unique microenvironments of oligolectic and polylectic provisions can preferentially filter microbes based on the nutritional chemistry of pollen and nectar, thereby shaping the community composition within the provisions . Indeed, recent findings indicate minimal overlap between the microbial communities associated with the pollen provisions of O. ribifloris and O. lignaria, the differences likely being driven by a combination of factors such as diet breath, local environment, and nest-building materials (Rothman et al., 2019(Rothman et al., , 2020.
Collectively, these studies suggest that both bee species host a taxonomically unique community of well-adapted microbes within their pollen provisions. Although comparing the microbial community associated with both bee species fell beyond the scope of this study, our findings reveal that notwithstanding their taxonomic specificity, the conspecific exosymbionts of both the polylege and oligolege are significant determinants of bee fitness outcome.
While our study offers compelling evidence supporting the function of exosymbiotic microbes in shaping bee fitness, a possible alternative explanation of our results is that the sterilization process may have compromised the nutritional value of pollen, confounding our findings. However, past studies did not find any significant difference between the nutrient profile of sterilized versus unsterilized pollen Dharampal, Hetherington, et al., 2020). Another study using laboratory-reared bumblebees showed significant colony growth when fed sterilized pollen that was recolonized by non-pathogenic microbes (Steffan et al., 2017). This suggested that the sterilization technique itself, did not produce any measurable adverse effect on pollen nutrient composition or the fitness outcome of bees that consumed pollen sterilized in this manner. Thus, based on prior research and direct quantification, it is unlikely that the sterilization of pollen resulted in a marked decline in diet quality, leading to the trends reported here. Another potential limitation of our study is that we did not investigate the extent to which larval digestive physiology may have influenced our results.
The ability of larval bees to digest different pollen types may depend on their metabolic capabilities. However, if larval nutrition was solely driven by their intrinsic metabolic capacity to digest pollen, it would not explain the dramatic mortality among larvae that were offered ample amounts of conspecific-sourced pollen, but not the pollen-associated microbiota. Another facypossible limitation of our study is that we did not ascertain the extent of microbial recolonization in Study 2. Our data reveal that larvae reared on sterilized diets, which were inoculated with microbes, showed high survivorship compared to those on sterilized diets, which were not inoculated. Thus, the difference in bee survival was likely mediated by the symbiotic pollen-associated microbes and this was strongly indicative of successful microbial recolonization of the sterilized pollen. We also acknowledge that our study investigated a single representative species of both foraging strategies among solitary bees. Moreover, since our study design required a large number of bees for adequate replication, we elected to use the significantly more abundant male progeny. Given the differences in life history traits, it would be interesting to investigate whether our findings would vary based on gender. Further studies using males and females from additional representative oligolectic and polylectic species will be needed to establish the potential functions of conspecific microbiota for the bee species hosting them.
The symbioses between bees and microbes represent one of the major paradigms in insect-microbe interactions. Yet, the relationship between solitary bees and their exosymbionts has remained poorly resolved. Our study contributes to this existing knowledge gap by demonstrating that the identity of microbial symbionts within pollen provisions is just as critical for larval development as the pollen source itself. Findings presented here indicate that the appropriate pairing of conspecific-sourced microbes with conspecific-sourced pollen yields the greatest benefit for developing solitary bees than either component by itself-a phenomenon that appears to be consistent across oligoleges and polyleges alike. This represents strong evidence that there is some degree of specificity between a given bee species, its particular pollen diet, and the natural microbiota therein. Thus, if there are disruptions to this innate coupling via external stressors, it could cause severe declines in bee fitness. For instance, exposure to fungicides during foraging trips can contaminate larval pollen provisions, leading to elevated concentrations of fungicide residues within nest-stored pollen (Artz & Pitts-Singer, 2015;Sgolastra et al., 2017Sgolastra et al., , 2018. This can cause detrimental alterations to the symbiotic microbial community by removing beneficial taxa and/or by increasing susceptibility to opportunistic pathogens (Steffan et al., 2017). Given that our study identifies pollen-associated microbiota as being just as important as the identity of the forage pollen itself, conserving the partnership between bees and their exosymbionts will be critical for maintaining healthy bee populations.

ACK N OWLED G M ENTS
We thank Kimball Clark and Dave Hunter for providing bee nesting reeds, Dr. Jun Zhu for assistance with statistical analysis, and Nolan Amon and Brandon Gominho for providing helpful suggestions on manuscript drafts. Molly Bidwell and John Lake provided laboratory assistance.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.